Ancient marine sediment DNA reveals diatom transition in Antarctica

Antarctica is one of the most vulnerable regions to climate change on Earth and studying the past and present responses of this polar marine ecosystem to environmental change is a matter of urgency. Sedimentary ancient DNA (sedaDNA) analysis can provide such insights into past ecosystem-wide changes. Here we present authenticated (through extensive contamination control and sedaDNA damage analysis) metagenomic marine eukaryote sedaDNA from the Scotia Sea region acquired during IODP Expedition 382. We also provide a marine eukaryote sedaDNA record of ~1 Mio. years and diatom and chlorophyte sedaDNA dating back to ~540 ka (using taxonomic marker genes SSU, LSU, psbO). We find evidence of warm phases being associated with high relative diatom abundance, and a marked transition from diatoms comprising <10% of all eukaryotes prior to ~14.5 ka, to ~50% after this time, i.e., following Meltwater Pulse 1A, alongside a composition change from sea-ice to open-ocean species. Our study demonstrates that sedaDNA tools can be expanded to hundreds of thousands of years, opening the pathway to the study of ecosystem-wide marine shifts and paleo-productivity phases throughout multiple glacial-interglacial cycles.


March 2021
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Study description
Research sample Sampling strategy The following databases were used in this study: SILVA small (version 132Ref-nr) and large (version 132Ref) subunit ribosomal RNA database (https://www.arbsilva.de/), and psbO53 (https://www.ebi.ac.uk/biostudies/studies/S-BSST659?query=S-BSST659). Source data are provided with this paper. Detailed Supplementary Information on methods and analysis is provided with this submission. The demultiplexed raw sequencing data generated and analysed during this study have been deposited in the NCBI Sequence Read Archive database (https://www.ncbi.nlm.nih.gov/sra) under Accession code/BioProject PRJNA861836 (https:// www.ncbi.nlm.nih.gov/bioproject/PRJNA861836/; BioSamples SAMN29928044 -SAMN29928123), and includes metadata for each sediment and control sample. For further requests please contact the corresponding author.
We present authenticated (through extensive contamination control and sedaDNA damage analysis) metagenomic marine eukaryote sedaDNA from the Scotia Sea region acquired during IODP Expedition 382. We also provide a marine eukaryote sedaDNA record of~1 Mio. years and diatom and chlorophyte sedaDNA dating back to~540 ka (using taxonomic marker genes SSU, LSU, psbO). We find evidence of warm phases being associated with high relative diatom abundance, and a marked transition from diatoms comprising <10% of all eukaryotes prior to~14.5 ka, to~50% after this time, i.e., following Meltwater Pulse 1A, alongside a composition change from sea-ice to open-ocean species. Our study demonstrates that sedaDNA tools can be expanded to hundreds of thousands of years, opening the pathway to the study of ecosystem-wide marine shifts and paleo-productivity phases throughout multiple glacialinterglacial cycles. The study is based on a total of 80 sedaDNA extracts/metagenomic shotgun libraries from 65 sediment samples, 6 air controls, 3 PFMD controls, and 6 extraction blank controls (see Data collection below).
Marine sediments collected via deep ocean sediment coring (see Sampling Strategy below). These samples include the ancient DNA of bacteria, archaea and eukaryota; the eukaryote portion is the focus of this research, they were not manipulated other than applying a sedaDNA extraction protocol that is optimised for the extraction of marine eukaryote sedaDNA (see Data collection below). The sediment samples are dated from present to~1 million years (see Dating methods below).

Sediment samples were collected during IODP Exp. 382 'Iceberg Alley and Subantarctic Ice and Ocean Dynamics' on-board RV Joides
Resolution between 20 March and 20 May 2019. Specifically, we collected samples at Site U1534 (Falkland Plateau, 606 m water depth), U1536 (Dove Basin, Scotia Sea, 3220 m water depth), and Site U1538 (Pirie Basin, Scotia Sea, 3130 m water depth). We used advanced piston coring (APC) to acquire sediment cores, which recovers the least disturbed sediments. All sediment samples were taken on the ship's 'catwalk', where, once the core was on deck, the core liners were wiped clean twice (3% sodium hypochlorite, 'bleach') at each cutting point. Core cutting tools were sterilised before each cut (3% bleach and 80% ethanol) of the core in 1m sections. The outer~3 mm of surface material were removed from the bottom of each core section to be sampled, using sterilised scrapers (~4 cm wide; bleach and ethanol treated). A cylindrical sample was taken from the core centre using a sterile (autoclaved) 10 mL cut-tip syringe, providing~5 cm3 of sediment material. The syringe was placed in a sterile plastic bag (Whirl-Pak) and immediately frozen at !80°C. The mudline (sediment/seawater interface) was transferred from the core liner into a sterile bucket (3% bleach treated), and 10 mL sample was retained in a sterile 15 mL centrifuge tube (Falcon) and frozen at !80°C. Samples were collected at various depth intervals depending on the site to span the Holocene up to~1 million years (Table 1). This lower depth/age limit was determined by switching coring system from APC to the extended core barrel (XCB) system. To test for potential airborne contamination, two air control was taken during the sedaDNA sampling process per site. For this, an empty syringe was held for a few seconds in the sampling area and then transferred into a sterile plastic bag and frozen at -80°C. In total, we collected 65 sediment samples, and 6 air controls.
To assess the potential for contamination due to drill fluid making contact with the core liner, we added the non-toxic chemical tracer perfluoromethyldecalin (PFMD) to the drill fluid at a rate of~0.55 mL min-1 for cores collected at Sites U1534 and U1536. As we found that PFMD concentrations were very low at these sites, the infusion rate was doubled prior to sedaDNA sampling at Site U1538 to ensure low PFMD concentrations represent low contamination and not delivery failure of PFMD to the core. At each sedaDNA sampling depth, one PFMD sample was taken from the periphery of the core (prior to scraping, to test whether drill fluid reached the core pipe), and one next to the sedaDNA sample in the centre of the core (after scraping, to minimise differences to the sedaDNA sample, and testing if drill fluid had reached the core centre). We transferred~3 cm3 of sediment using a disposable, autoclaved 5 mL cut-tip syringe into a 20 mL headspace vial with metal caps and Teflon seals. We also collected a sample of the tracer-infused drill fluid at each site, by transferring~10 mL of the fluid collected at the injection pipe on the rig floor via a sterile plastic bottle into a 15 mL centrifuge tube (inside a sterile plastic bag) and freezing it at -80°C. Samples were analysed using gas chromatography (GC-µECD; A detailed description of the PFMD GC measurements is provided in Weber et al., Proceedings of the IODP Exp 382, 2021. Briefly, PFMD measurements were undertaken in batches per site for U1534, U1536, and U1538. This included the analyses of PFMD samples collected at two additional holes at these sites, U1534D and U1536C, from which we also collected sedaDNA samples but that are not part of this study. PFMD is categorized as the stereoisomers of PFMD (C11F20), which add up to 87-88% (and with the remaining 12% being additional perfluoro compounds unable to be separated by the manufacturer). We exclusively refer to the first and measurable PFMD category, calibrating for the 88% in bottle concentrations during concentration calculations. Each GC analysis run included the measurement of duplicate blanks and duplicate PFMD standards. Due to a large sample number, PFMD at Site U1538 was measured in three separate runs, with the first and last run including triplicate blank and triplicate PFMD standards (duplicates in the second run), and the last run also containing a drill-fluid sample. To blank-correct PFMD concentrations, we subtracted the average PFMD concentration of all blanks per run from PFMD measurements in that run. To determine the detection limit of PFMD, we used three times the standard deviation of the average blank PFMD values per run; due to all blank values for the U1538 runs being 0, we used three times the standard deviation of the lowest PFMD standard for this site in this calculation. This provided us with a PFMD detection limit of 0.2338 ng mL-1. Any PFMD measurements of samples below this limit were rejected.
A total of 80 sedaDNA extracts and metagenomic shotgun libraries were prepared following Armbrecht et al., MER, 2020, andArmbrecht et al. Sci.Rep., 2021. This included 65 sediment samples, 6 air controls, 3 PFMD controls, and 6 extraction blank controls (see below). We randomised our samples and controls and extracted sedaDNA in batches of 16 extracts/libraries at a time, with each batch including at least one air control and one extraction blank control (EBC), and the last batch including mudline and PFMD samples to avoid contamination of the sedaDNA samples. A complete list of sedaDNA samples and controls is provided in the manuscript ( Table 1). The laboratory procedure was documented by the corresponding author in digital and paper-based protocols and notes. The libraries sequenced at the Garvan Institute for Medical Research, Sydney, Australia (Illumina NovaSeq 2 x 100 bp). PFMD data: Any PFMD measurements of samples below the detection limit (0.2338 ng mL-1) were rejected/excluded. sedaDNA data: Reads for species identified in EBCs, air, and PFMD controls were subtracted from sediment sample setaDNA data, which was conducted with MEGAN CE (version 6.21.12). All taxa determined in the controls are provided with the Supplementary Data.
Our dataset is unique, and was generated over a total of 3 years including sediment coring of the deep seafloor in remote Antarctica, laboratory work, and bioinformatic processing, and analysis. It was generated using the most stringent, recommended anticontamination procedures. During the laboratory phase samples were randomised, and 20% of the dataset included controls. Therefore, we have not repeated this study.
We randomised our samples and controls and extracted sedaDNA in batches of 16 extracts/libraries at a time, with each batch including at least one air control and one extraction blank control (EBC), and the last batch including mudline and PFMD samples to avoid contamination of the sedaDNA samples.
During the sedaDNA extraction and library preparation process, we labelled the samples with running numbers from 1-16 (16 samples per batch). This means there were no biases as to what sample was a sediment sample, control, specific site, or specific age. Bioinformatic processing was performed the same way for all samples and controls using the same commands, thus biases in the computational analyses were impossible.
Field work included sediment coring of the deep ocean in remote Antarctica. Generally, sediment coring is only possible in relatively calm seas. Upon retrieval, sediment cores were immediately transported to an indoors area (the 'catwalk') nearby, where core cutting and sampling under clean conditions (described above) took place. The catwalk is open to the outdoors at one end, i.e., sampling occurred under normal, cool air conditions characteristic for the study region, with no exposure to precipitation.
IODP Expedition proposals undergo a rigorous environmental protection and safety review, which is approved by the IODP's Environmental Protection and Safety Panel (EPSP) and/or the Safety Panel. The same procedure was applied to IODP Exp. 382 and approval was provided by the EPSP. Sediment samples for sedaDNA analyses were imported to Australia under Import Permit number 0002658554 provided by the Australian Government Department for Agriculture and Water Resources (date of issue: 19 September 2018), and were stored and extracted at a quarantine approved facility (AA Site No. S1253, Australian Centre for Ancient DNA).
Neither fieldwork, lab work or bioinformatic data processing caused any disturbances.